Glycans are complex, heterogenous biological sugar polymers generally found attached to protein or lipids and are displayed on cell and macromolecular surfaces. Glycans are created in the endoplasmic reticulum and golgi apparatus organelles by enzymes known as glycotransferases. The molecular structure of glycans strongly affects the biological activity of the protein or lipid to which it is attached. Aberrant glycotransferase activity (which, in turn, produces aberrant glycan structures) is a well-known hallmark of essentially every known type of cancer.
The altered expression of a single glycotransferase can result in the production of a complex, heterogeneous mixture of n unique, abnormal whole-glycan structures rather than in an uniformly increase expression of a single whole-glycan structure (FIG. 1). These heterogeneous mixtures of whole-glycan structures are often very difficult to fully characterize. Thus, existing cancer biomarkers that are based on intact glycan structure are generally based on one or a few particular aberrant glycan structures (out of n) or perhaps as set of very closely related aberrant glycan structures that result in a unique antibody or lectin epitope.
One known method for characterizing glycans on the basis of molecular weight consists of permethylation, release from the protein substrate, and analysis intact by mass spectrometry. In this approach, a pre-isolated glycan or pre-isolated O-linked glycoprotein is permethylated, purified and analyzed intact by MALDI-mass spectrometry (MS) or liquid chromatography (LC)-electrospray ionization (ESI)-MS. Goetz et al. (“Enzymatic/Chemical Release of O-Glycans Allowing MS Analysis at High Sensitivity.” Anal. Chem., 2009, 81 (23), pp 9546-9552) describe this method. Importantly, Goetz et al. state that their method does not release N-linked glycans from proteins and they did not discuss glycolipids in this paper. The analysis of N-linked glycans by this approach would require preliminary cleavage from their protein substrates (usually by enzyme), followed by isolation then permethylation. Most notably, Goetz et al. describe their method as a means to analyze pre-isolated glycoproteins or glycans. They do not consider complex mixtures of glycoproteins, whole biofluids or tissues.
A second known method for characterizing pre-isolated glycans known as glycan methylation analysis was developed by the University of Georgia's Complex Carbohydrate Research Center (CCRC) [Heiss et al. (“The structure of Cryptococcus neoformans galactoxylomannan contains beta-D-glucuronic acid,” Carbohydrate Research 2009, 344(7), pp 915-20)]. This method is a trifluoroacetic acid (TFA)-based methylation analysis and consists of permethylation, hydrolysis, reduction and acetylation